Cell therapy by graft of autologous or nonautologous cells constitutes a major therapeutic tool which is at present essentially developed in hemobiology, but should be applicable to other specialties based on the knowledge acquired regarding stem cells and their identification in most tissues, ranging from muscle to the central nervous system. The increasing identification and characterization of cytokines and growth factors allow us to envisage the possibility of in vitro and/or in vivo control of the proliferation and differentiation of these cells and the modulation of their tissue environment (immunologic rejection phenomena, angiogenesis). Despite these advances in cell biology, the clinical development of cell grafts remains limited at present, notably because of the low survival rate of the implanted cells which can be linked to a nonspecific mortality (cell death by necrosis or apoptosis) due to the procedures employed for collection, storage, transformation and administration or to an immunologic rejection (in allografts and xenografts), i.e., the absence of integration in the host tissue.
It has been proposed to use nonbiodegradable microbeads on which the cells adhere thereby functioning as transporters or microcarriers to reduce this cellular mortality. For example, the survival and functioning of hepatocytes were improved when such cells were grafted/adhered to glass or dextran (Cytodex®) microbeads (Demetriou et al., 1986; Te Velde et al., 1992). This strategy has made it possible to obtain more promising results than with microencapsulated hepatocytes.
More recently, these same microbeads have been used for cultivating and grafting human keratinocytes to reconstitute a cutaneous cover in the nude mouse (Voigt et al., 1999). This approach has also been used for grafting neurochromaffins or dopaminergic embryonic neurons in a murine model of Parkinson's disease. In this model, the survival of the transplanted cells in the striatum is greatly increased when they are first adhered to glass or dextran microparticles, thereby enabling behavioral improvement of the animals (Cherskey et al., 1996; Saporta et al., 1997; Borlongan et al., 1998).
It has also been observed (Saporta et al., 1997) that human fetal cells adhered on dextran microbeads survive for at least three months without immunosuppressant treatment, whereas such cells without microparticles are rapidly rejected.
Another more recent approach enabling augmentation of the survival of grafted cells is the administration of growth factors in association with the graft. These proteins, which can act on proliferation, differentiation, activation and survival of the cells, constitute a major contribution to the field of cell grafts. Although it is now possible to have available human recombinant growth factors, their administration represents a challenge because these products have a short half-life and do not cross certain biological barriers. They moreover have a pleiotropic action which can be the cause of undesirable side effects. The presently developed modes of administration are not completely satisfactory and/or applicable in clinical practice.
One of the first modes of administration proposed grafting cells in a suspension containing growth factor. Although this approach is simple, it does not enable long-term action on the cells. A second mode of administration consists of co-transplanting a tissue identified as producing the selected growth factor, e.g., peripheral nerve-chromaffin cell co-grafts (Date et al., 1996) or hepatocyte-islets of Langerhans co-grafts (Kneser et al., 1999). The sometimes limited survival of such co-grafts and the inability to control the doses of growth factors considerably limits this strategy. Progress made in molecular biology now allows for the production of genetically modified cells producing a growth factor which can be used in co-grafts or in grafts as usually defined (Menei et al., 1998; Wood and Prior, 2001). Nevertheless, this approach remains limited by ethical problems, biological risk and control of the released doses. Grafts of nerve cells have been reported such as PC12 neuroendocrine cells (Menei et al., 1989; Dehaut et al., 1993), and normal Schwann cells or Schwann cells genetically modified to produce a neurotrophic factor (Montero-Menei et al., 1992; Menei et al., 1998).
There have also been reports of biodegradable microparticles releasing neuroactive molecules in a controlled and prolonged manner (Menei et al., 1997; Benoit et al., 1999). These microspheres are constituted of a biopolymer of the poly(lactic acid-glycolic co-acid) (PLGA) type. They are biocompatible with nerve tissue and totally degraded in several months (Menei et al., 1993; 1994b; Véziers et al., 2000). Their size of several tens of microns allows stereotactic implantation in the brain at the level of their pharmacological target using the same microsyringes as for cell implantations (Menei et al., 1994a). They were used successfully in a phase I clinical study for the interstitial chemotherapy of brain tumors (Menei et al., 1999).
Microspheres releasing proteins, in particular growth factors and cytokines, have also been developed. Nerve growth factor (NGF) is a substance of interest because it was among the earliest characterized. There have been descriptions of microspheres that can release NGF over at least two months (Péan et al., 1998; Péan et al., 1999). Their therapeutic value was demonstrated on two animal models of neurodegenerative diseases: the murine model of Alzheimer's disease (Péan et al., 2000) and the murine model of Huntington's chorea (Menei et al., 2000).
In the field of tumors, PLGA microspheres have been formulated which are capable of releasing immunostimulant cytokines after intratumoral implantation (Mullerad et al. 2000; Pettit et al., 1997). The use of biodegradable microspheres for the release of cytokines in the framework of antitumor vaccine was therefore proposed (Golumbek et al., 1993). However, in that study the microspheres were simply mixed with the cells immediately prior to injection. In fact, the preparation of vaccine constituted of microspheres coated by bacterial antigens or membrane vesicles had already been proposed, but without the microspheres having the ability to release immunostimulant molecules (Mescher and Rogers, 1996; Mesher and Savelieva, 1997).